Elevated mucus secretion and accumulation is the hallmark of airway diseases. This aberrance is manifested by mucous cell metaplasia. Evidences have shown that mucous cell metaplasia is the consequence of an intrinsic increase of mucin gene expression. Through a microarray gene expression profile study of retinoic acid-induced airway epithelial cell differentiation, we have discovered that a small GTP binding protein, Rad, plays an important role in the attenuation of mucin gene expression mad secretion. Immunohistchemical studies of human tracheobronchial and esophageal tissues indicated that Rad protein is abundant in ciliated and squamous cells, but scarce in mucous cells. Both Northern blot and RealTime RT-PCR analysis demonstrated that the steady state level of MUC5B/MUC5AC mRNA were drastically inhibited by Rad. Co-transfecfion of mucin gene promoter-reporter gene consh,"oct and Rad showed that the promoter activities of MUC5B/5AC were attenuated by Rad. Interestingly, when both C terminal and N terminal extension of Rad gene were deleted, and the GTP-binding site of Rad at Ser66 was mutated, the effect of Rad on mucin gene expression was reversed. Dual immuno-fluorescent staining indicated that Rad protein is co-localized with B-tubulin in microtubule. All of the above results strongly suggested that Rad could be a "master switch" factor governing mucous cell process via its interaction with microtubule. In this proposal, we hypothesize that Rad protein regaalates MUC gene expression by attenuating MUC5B/MUC5AC promoter activities. In order to test the hypothesis and to develop the Rad based molecular approach as a potential therapeutic tool, the following specific aims are proposed: 1) To determine the transcription factor-DNA binding site and the transcription factors involved in the Rad mediated signaling attenuation of MUC gene expression. 2) To elucidate the structure-function relationship of Rad in the attenuation of MUC5B gene expression. 3) To develop a biological paradigm for primary human airway epithelial cells and in vivo studies of the function of Rad in the attenuation of MUC gene expression. These to-be-obtained results will not only be of interest to general biological science, but also assist us to develop a potential effective therapeutic tool for the control of mucin production. In summary, we have proposed a comprehensive approach utilizing combined in vitro, cell, and in vivo methods to test our hypothesis.